Compositions and methods for decreasing, or preventing or reversing gain of, skin pigmentation in a mammalian subject

ABSTRACT

The present invention includes compounds, compositions comprising the same, and methods using the compounds and/or compositions therein, for modulating skin pigmentation in a mammalian subject. In certain embodiments, the compounds of the invention treat or prevent skin disorders or diseases associated with hyperpigmentation in the subject. In other embodiments, the compounds of the invention act as antagonist to the non-canonical sex steroid hormone receptor GPRE1 and do not bind to a canonical nuclear estrogen receptor (ER). In yet other embodiments, the compounds of the invention comprise acts as agonists to the non-canonical sex steroid hormone receptor PAQR7 and do not bind to a canonical nuclear progesterone receptor (PR).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. § 371 national phase applicationfrom, and claims priority to, International Application No.PCT/US2016/026213, filed Apr. 6, 2016, and published under PCT Article21(2) in English, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Applications No. 62/143,597, filed Apr. 6, 2015, and No.62/216,792, filed Sep. 10, 2015, all of which applications areincorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA 163566 awardedby National Institutes of Health/National Cancer Institute, and underCA174523 awarded by National Institutes of Health SPORE. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Human skin color ranges in variety from the darkest brown to thelightest pinkish-white hues. Skin pigmentation in humans evolvedprimarily to regulate the amount of ultraviolet (UV) radiationpenetrating the skin, thus controlling the radiation's biochemicaleffects. The most important substance that affects the actual skin colorof humans is the pigment melanin.

Melanin controls the amount of UV radiation from the sun that penetratesthe skin by absorption. While UV radiation can assist in the productionof vitamin D, excessive exposure to UV can damage health. Melanin isproduced within the skin in cells called melanocytes, and is the maindeterminant of the skin color of darker-skinned humans. Both the amountand type of melanin produced is controlled by a number of genes thatoperate under incomplete dominance. Melanocytes residing in the basalepidermis control human skin color through synthesis of melanin(melanogenesis), a complex process thought to be primarily regulated byalpha-melanocyte stimulating hormone (αMSH). The αMSH peptide issecreted centrally by the pituitary, and locally by surroundingkeratinocytes in response to UV damage. αMSH binding to the melanocortinreceptor 1 (MC1R), a G protein-coupled receptor (GPCR), activatesadenylate cyclase, and increases cytosolic cAMP. This secondarymessenger activates a cascade of downstream transcriptional eventsleading to expression of genes required for melanin synthesis.

Melanogenesis takes place within small membrane-bound packages calledmelanosomes. As the melanosomes become full of melanin, they move intothe slender arms of melanocytes, from where they are transferred to thekeratinocytes. Under normal conditions, melanosomes cover the upper partof the keratinocytes and protect them from genetic damage. Onemelanocyte supplies melanin to about 36 keranocytes according to signalsfrom the keranocytes. They also regulate melanin production andreplication of melanocytes. People have different skin colors mainlybecause their melanocytes produce different amount and kinds of melanin.The genetic mechanism behind human skin color is mainly regulated by theenzyme tyrosinase, which creates the color of the skin, eyes, and hairshades. Differences in skin color are also attributed to differences insize and distribution of melanosomes in the skin.

Uneven pigmentation of some sort affects most people, regardless ofbioethnic background or skin color. Skin may appear lighter or darkerthan normal, or lack pigmentation at all. There may be blotchy anduneven areas, patches of brown to gray discoloration, or freckling.Apart from blood-related conditions such as jaundice, carotenosis, orargyria, skin pigmentation disorders generally occur because the bodyproduces either too much or too little melanin.

There is a need in the art to develop novel compositions and methods fordecreasing, or preventing or reversing gain of, skin pigmentation in amammalian subject, such as a human. Such compositions and methods wouldbe useful, in non-limiting embodiments, for treating or preventing skindisorders or diseases associated with hyperpigmentation. Suchcompositions and methods would be useful, in non-limiting embodiments,for treating or preventing skin disorders or diseases associated withpregnancy. The present invention fulfills this need.

BRIEF SUMMARY OF THE INVENTION

The invention provides a compound, or a salt, solvate, tautomer,enantiomer or diastereoisomer thereof. The invention further provides apharmaceutical composition comprising at least one pharmaceuticallyacceptable carrier and at least one compound of the invention. Theinvention further provides a method of decreasing, or preventing orreversing gain of, skin pigmentation in a mammalian subject. Theinvention further provides a kit for decreasing, or preventing orreversing gain of, skin pigmentation in a mammalian subject.

In certain embodiments, the compound or molecule of the invention is

wherein: R₁ is selected from the group consisting of H and C₁-C₃ alkyl;R₂ is selected from the group consisting of H and C₁-C₃ alkyl; bond α isa single or double bond; R₃ is selected from the group consisting of Hand halo.

In certain embodiments, the compound or molecule, or a salt, solvate,tautomer, enantiomer or diastereoisomer thereof, is G-15(rel-(3aS,4S,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline).In other embodiments, the compound or molecule, or a salt, solvate,tautomer, enantiomer or diastereoisomer thereof, is not G-15.

In certain embodiments, the compound or molecule, or a salt, solvate,tautomer, enantiomer or diastereoisomer thereof, is G-36(rel-(3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-8-isopropyl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolone).In other embodiments, the compound or molecule, or a salt, solvate,tautomer, enantiomer or diastereoisomer thereof, is not G-36.

In certain embodiments, the compound or molecule, or a salt, solvate,tautomer, enantiomer or diastereoisomer thereof, is CH2P4(rel-(8S,9S,10S,13S,14S,17S)-17-acetyl-13-methyl-10-vinyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3(2H)-one).In other embodiments, the compound or molecule, or a salt, solvate,tautomer, enantiomer or diastereoisomer thereof; is not CH2P4.

In certain embodiments, in (I) R₁ is H or 2-propyl. In otherembodiments, in (I) R₂ is H or methyl. In yet other embodiments, in (I)bond α is a double bond. In yet other embodiments, in (I) bond α is asingle bond. In yet other embodiments, R₃ is selected from the groupconsisting of H, F, Cl, and Br.

In certain embodiments, the compound is at least one selected from thegroup consisting of:

In certain embodiments, the pharmaceutical composition is formulated fortopical or transdermal administration. In other embodiments, thepharmaceutical composition does not comprise G-15. In yet otherembodiments, the pharmaceutical composition does not comprise G-36. Inyet other embodiments, the pharmaceutical composition does not compriseCH2P4. In yet other embodiments, the pharmaceutical composition furthercomprises at least one sun-blocking agent. In yet other embodiments, thepharmaceutical composition further comprises at least one sunscreenlotion.

In certain embodiments, the method comprises administering to thesubject a therapeutically effective amount of at least one compoundselected from the group consisting of: (i) a GPER antagonist that doesnot bind to a canonical nuclear estrogen receptor, and (ii) a PAQR7agonist that does not bind to a canonical nuclear progesterone receptor.

In certain embodiments, the subject is human.

In certain embodiments, the canonical nuclear estrogen receptorcomprises at least one selected from the group consisting of ERα andERβ.

In certain embodiments, the canonical nuclear progesterone receptorcomprises at least one selected from the group consisting of PR-A andPR-B.

In certain embodiments, the compound is a GPER1 antagonist.

In certain embodiments, the subject is suffering from at least onecondition selected from the group consisting of pigmentary changesassociated with oral contraceptive use, pregnancy, and endogenousestrogens in females; solar lentigo; acne; eczema; chemical, sun, andthermal burn scars; lupus; psoriasis; sarcoidosis; pityriasis; erythemadyschromicum perstans; blistering diseases; drug reactions; lichenplanus; and other inflammatory skin insults.

In certain embodiments, the compound is administered topically ortransdermally to the subject. In yet other embodiments, the compound isformulated as a pharmaceutical composition for topical or transdermaladministration. In yet other embodiments, the pharmaceutical compositionfurther comprises at least one sun-blocking agent. In yet otherembodiments, the pharmaceutical composition further comprises asunscreen lotion. In yet other embodiments, the pharmaceuticalcomposition is essentially free of a skin bleaching agent.

In certain embodiments, the compound is at least one selected from thegroup consisting of G-15, G-36 and CH2P4.

In certain embodiments, the kit comprises at least one compound and/orpharmaceutical composition of the invention. In other embodiments, thekit further comprises instructions for topically or transdermallyadministering the at least one pharmaceutical composition to thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 comprises a set of images illustrating pregnancy-associatedpigmentation disorder. Images A-C depict pigmentation disorder ofpatients with melasma. Image D depicts a patient with pigmentarydemarcation lines. Images E and F depict typical linea nigra on amother. Image G depicts typical linea nigra on a child.

FIGS. 2A-2G comprise a set of scheme and bar graphs illustrating melaninproduction in human melanocytes. FIG. 2A comprises a schematicillustration of classical regulation of human melanocyte homeostasis.FIG. 2B comprises a bar graph illustrating melanin production inresponse to αMSH in a dose dependent manner. FIG. 2C comprises a bargraph illustrating melanin production in response to estrogen in a dosedependent manner. FIG. 2D comprises a bar graph illustrating melaninproduction by melanocytes treated with vehicle, αMSH, or estrogen. FIG.2E comprises a bar graph illustrating melanin production in response toprogesterone (P4) in a dose dependent manner. FIG. 2F comprises a bargraph illustrating melanin production in response to estrogen andprogesterone using iPS-derived female melanocytes. FIG. 2G comprises abar graph illustrating melanin production in response to estrogen andprogesterone using facial, aged-adult melanocytes. N=3 biologicreplicates for each experiment. Error bars denote ±s.d., *p<0.05.

FIGS. 3A-3F comprise a set of images and bar graphs illustrating thefinding that estrogen and progesterone reciprocally regulate melaninsynthesis. FIG. 3A comprises a bar graph illustrating melanin content ofprimary human melanocytes treated with estrogen (E2), compared tovehicle-treated controls. FIG. 3B comprises a bar graph and imagesillustrating Fontana-Masson (melanin) staining of organotypic skintreated with vehicle or estrogen. Relative melanin content isillustrated in the bar graph at the bottom of the Figure. FIG. 3Ccomprises a bar graph and images illustrating MITF(microphthalmia-associated transcription factor) immunohistochemistry oforganotypic skin treated with vehicle or estrogen. Melanocyte populationdensity is illustrated in the bar graph at the bottom of the Figure.FIG. 3D comprises a bar graph illustrating melanin content of primaryhuman melanocytes treated with progesterone (P4), compared to vehicle.FIG. 3E comprises a bar graph and images illustrating Fontana-Masson(melanin) staining of organotypic skin tissues treated with progesteroneor vehicle. Relative melanin content is illustrated in the bar graph atthe bottom of the Figure. FIG. 3F comprises a bar graph and imagesillustrating MITF immunohistochemistry of organotypic skin tissuestreated with vehicle or progesterone. Melanocyte population density isillustrated in the bar graph at the bottom of the Figure. N=3 biologicreplicates for each experiment. Error bars denote ±s.d., *p<0.05, scalebar=50 μm.

FIGS. 4A-4H comprise a set of images and bar graphs illustrating thefinding that estrogen and progesterone access the classical melaninproduction pathway through nonclassical receptors. FIG. 4A comprises abar graph illustrating cAMP ELISA from estrogen-treated melanocytes.FIG. 4B comprises a set of western blot images demonstrating changes inclassical melanin pathway regulators after estrogen treatment. FIG. 4Ccomprises a bar graph illustrating cAMP ELISA from progesterone-treatedmelanocytes. FIG. 4D comprises a set of western blot imagesdemonstrating changes in classical melanin pathway regulators afterprogesterone treatment. FIG. 4E comprises a bar graph illustrating amelanin assay from melanocytes treated with estrogen and progesteronesimultaneously. FIG. 4F comprises a set of western blot images forestrogen and progesterone receptors in MCF7 cells and melanocytes. FIG.4G comprises a bar graph illustrating melanin content of melanocytestransduced with control shRNA or shRNA targeting GPER. Cells weretreated with either vehicle or estrogen. FIG. 4H comprises a bar graphillustrating a melanin assay performed on melanocytes transduced withcontrol shRNA or shRNA targeting PAQR7. Cells were treated with eithervehicle or progesterone. N=3 biologic replicates for each experiment.Error bars denote ±s.d., *p<0.05.

FIGS. 5A-5H comprises a set of bar graphs and table illustrating hormonereceptors in melanocytes. FIG. 5A comprises a bar graph illustratingrelative gene expression of classical hormone receptors in MCF7 cellsand melanocytes, as determined by qRT-PCR. Ct values were normalized toactin, and set relative to the expression of androgen receptor (AR) inMCF7 cells. FIG. 5B comprises a table illustrating RPKM values forclassical and nonclassical estrogen and progesterone receptors in humanmelanocytes, by convention, RPKM values >1 indicate the gene isexpressed. FIG. 5C comprises a bar graph illustrating expression of GPERand Progestin and AdipoQ Receptor 7 (PAQR7) displayed as 1/Ct value.FIG. 5D comprises a bar graph illustrating relative expression of GPERand PAQR7 transcripts in melanocytes, fibroblasts, and keratinocytes, asdetermined by qRT-PCR, displayed relative to the expression level inmelanocytes. FIG. 5E comprises a bar graph illustrating qRT-PCR showingmRNA knockdown efficiency of the two hairpins targeting GPER. FIG. 5Fcomprises a bar graph illustrating qRT-PCR showing mRNA knockdownefficiency of the two hairpins targeting PAQR7. FIG. 5G comprises a setof bar graphs illustrating melanin content of melanocytes transducedwith LentiCRISPRV2 with guide RNA targeting GFP or GPER. Cells weretreated with either vehicle or estrogen. FIG. 5H comprises a set of bargraphs illustrating melanin content of melanocytes transduced withLentiCRISPRV2 with guide RNA targeting GFP or PAQR7. Cells were treatedwith either vehicle or progesterone. Error bars denote ±s.d., *p<0.05.

FIG. 6 is a bar graph illustrating melanin synthesis in response toPertussis Toxin (PTX), progesterone (P4), or both. N=3 biologicreplicates for each experiment. Error bars denote ±s.d., *p<0.05.

FIGS. 7A-7B comprise a set of bar graphs illustrating the finding thatmelanin production is altered by sex steroid analogs—GPER agonistscurrently in clinical use. FIG. 7A is a bar graph illustrating melaninproduction in response to tamoxifen (TMX). FIG. 7B is a bar graphillustrating melanin production in response to ethinyl estradiol (EE2).N=3 biologic replicates for each experiment. Error bars denote ±s.d.,*p<0.05.

FIG. 8 comprises a bar graph illustrating melanin production bymelanocytes treated with vehicle, a GPER agonist, or estrogen, in thepresence of selective GPER antagonists G-15 or G-36. N=3 biologicreplicates for each experiment. Error bars denote ±s.d., *p<0.05.

FIGS. 9A-9B comprise a set of bar graphs illustrating the effects ofspecific agonists targeting PAQR7 on melanin production. FIG. 9Acomprises a bar graph illustrating melanin production in response toCH2P4, a specific PAQR7 agonist. FIG. 9B comprises a bar graphillustrating melanin assay performed on melanocytes lentivirallytransduced with control shRNA or shRNA targeting PAQR7. These cells weretreated with either vehicle or CH2P4. N=3 biologic replicates for eachexperiment. Error bars denote ±s.d., *p<0.05.

FIGS. 10A-10C comprise a set of images and bar graphs illustrating thefinding that GPER signaling is sufficient to alter melanin production inorganotypic human tissue. FIG. 10A comprises a set of imagesillustrating organotypic skin treated with vehicle (left) or CH2P4(right). FIG. 10B comprises a set of images and bar graph illustratingFontana-Masson (melanin) staining of organotypic skin treated withvehicle or CH2P4. Quantification of melanin content is illustrated onthe right. FIG. 10C comprises a set of images and bar graph illustratingMITF immunohistochemistry of organotypic skin treated with vehicle orCH2P4. Quantification of melanocyte population density is illustrated onthe right. N=3 biologic replicates for each experiment. Error barsdenote ±s.d., *p<0.05, scale bar=50 μm.

FIG. 11 comprises a schematic representation of selected biologicalpathways discussed herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in one aspect, to the unexpecteddiscovery that compounds of the invention modulate skin pigmentation ina mammalian subject, such as a human. In certain embodiments, thecompounds of the invention treat or prevent skin hyperpigmentation in asubject. In other embodiments, the compounds of the invention treat orprevent wrinkles and/or discolorations of premature aging. In yet otherembodiments, the compositions of the invention modulate skin color foresthetic benefit without exposure to skin bleaching products and/orDNA-damaging UV radiation. In yet other embodiments, the compositions ofthe invention help revert or minimize estrogen-associated skin changes(such as, for example, darkening of the skin, appearance of skin spots,and the like), which may take place for example during pregnancy and/orduring therapeutic treatment with estrogen or analogs thereof.

Human pregnancy and oral contraceptive use are commonly associated withaltered epidermal pigmentation, and this suggests that changes incirculating hormones may regulate melanocyte homeostasis. However, thespecific hormones, receptors, and downstream pathways mediating theseeffects have been undefined.

As demonstrated herein, physiologically relevant levels of 17β-estradiol(estrogen) promotes skin pigment production, while progesterone hasopposing effects. Altered melanin production is associated with parallelchanges in cAMP, a known regulator of mammalian pigmentation. Normalprimary melanocytes do not express the classical estrogen andprogesterone receptors (ER/PR). The present studies demonstrate thatmelanocytes express the non-canonical steroid hormone receptors Gprotein-coupled estrogen receptor 1 (GPER1), and Progestin and AdipoQReceptor 7 (PAQR7). Lentiviral-mediated CRISPR-Cas9 ablation of theseproteins blocks the pigmentary effects of estrogen and progesterone,suggesting that estrogen and progesterone reciprocally affectpigmentation through the non-canonical receptors GPER1 and PAQR7,respectively, to regulate human melanocyte homeostasis. In certainaspects, modulation of these receptors and their associated signalingpathway elements have therapeutic utility in disorders of epidermalpigmentation.

The results presented herein, utilizing both genetic depletion andspecific pharmacologic agonists and antagonists, establish that estrogenand progesterone influence human melanocyte pigment production throughactivation of the nonclassical hormone receptors GPER and PAQR7,respectively (FIG. 11). In one non-limiting aspect, specific activationof these receptors using selective agonist or antagonists is sufficientto increase or decrease pigment synthesis, avoiding the potentiallyundesirable effects of classical estrogen and progesterone receptoractivation in other cell types. GPER and PAQR7 have reduced expressionin keratinocytes or fibroblasts, which should limit drug effects to thecells of interest, in contrast to forskolin, which nonspecificallyactivates adenylate cyclase in all cells.

In non-limiting embodiments, G-15 and G-36 are GPER antagonists, and donot bind to a canonical nuclear ER. In certain embodiments, thecanonical nuclear ER comprises ERα and/or ERβ).

In non-limiting examples, CH2P4 is a PAQR7 agonist and does not bind toa canonical nuclear PR. In certain embodiments, the canonical nuclear PRcomprises PR-A and/or PR-B.

In one aspect, specific GPER antagonists and PAQR7 agonists represent auseful, novel class of therapeutics for normalizing disorders ofepidermal pigmentation. Myriad genetic and acquired conditions includingcommon afflictions, such as acne, eczema, vitiligo, ultraviolet (UV)radiation exposure, traumatic injury, and pregnancy, are associated withalterations in skin pigmentation that can be extensive and long-lasting.

In certain non-limiting embodiments, targeting the receptor(s) describedherein can protect skin from wrinkles, and discoloration of prematureaging, and also modulate skin color for esthetic benefit withoutexposure to DNA-damaging UV radiation or toxic skin bleaching agents.

The invention should not be construed to be limited to the compounds, orany analog thereof, recited herein. In certain embodiments, the presentinvention encompasses any compounds that act as antagonists towards GPERand do not bind to the canonical nuclear estrogen receptor. In otherembodiments, the present invention encompasses any compounds that act asagonists towards PAQR7 and do not bind to the canonical nuclearprogesterone receptor.

Definitions

As used herein, each of the following terms have the meaning associatedwith it in this section.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics and chemistry are those well-known andcommonly employed in the art.

As used herein, the term “19-CH2P4” or “CHP4” refers torel-(8S,9S,10S,13S,14S,17S)-17-acetyl-13-methyl-10-vinyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3(2H)-one),or a salt, solvate, enantiomer or diastereoisomer thereof.

As used herein, the articles “a” and “an” refer to one or to more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

As used herein, the term “αMSH” refers to alpha-melanocyte stimulatinghormone.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent on the context inwhich it is used. As used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, the term “about”is meant to encompass variations of ±20% or ±10%, ±5%, ±1%, or ±0.1%from the specified value, as such variations are appropriate to performthe disclosed methods.

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined elsewhere herein,connected to the rest of the molecule via an oxygen atom, such as, forexample, methoxy, ethoxy, 1-propoxy, 2-propoxy (or isopropoxy) and thehigher homologs and isomers. A specific example is (C₁-C₃)alkoxy, suchas, but not limited to, ethoxy and methoxy.

As used herein, the term “alkyl” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbon atoms) and includes straight, branched chain, orcyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. A specific embodiment is (C₁-C₄)alkyl, such as, butnot limited to, ethyl, methyl, isopropyl, n-butyl, isobutyl, t-butyl,and cyclopropylmethyl.

As used herein, the term “AR” refers to androgen receptor.

As used herein, the term “cAMP” refers to cyclic adenosinemonophosphate.

As used herein, the term “CREB” refers to cAMP response element-bindingprotein.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated, theanimal's health continues to deteriorate. In contrast, a “disorder” inan animal is a state of health in which the animal is able to maintainhomeostasis, but in which the animal's state of health is less favorablethan it would be in the absence of the disorder. Left untreated, adisorder does not necessarily cause a further decrease in the animal'sstate of health.

As used herein, the term “E2” refers to estrogen.

As used herein, the term “EE2” refers to ethinyl estradiol.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” or “pharmaceutically effective amount” of a compoundare used interchangeably to refer to the amount of the compoundsufficient to provide a beneficial effect to the subject to which thecompound is administered.

As used herein, the term “ER” refers to estrogen receptor.

As used herein, the term “GPCR” refers to a G protein-coupled receptor.

As used herein, the term “G15” or “G-15” refers torel-(3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-2,3,3a,4,5,9b-hexahydro-1H-cyclopenta[c]quinolone,or a salt, solvate, enantiomer or diastereoisomer thereof.

As used herein, the term “G36” or “G-36” refers torel-(3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-8-isopropyl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolone),or a salt, solvate, enantiomer or diastereoisomer thereof.

As used herein, the term “GPR30 receptor” (also known as GPER1; CEPR;CMKRL2; DRY12; FEG-1, GPCR-Br; GPER; LERGU; LERGU2; LyGPR; or mER)refers to a 7-transmembrane (7TM) G protein-coupled receptor thatmediate estrogen-dependent signal transduction. GPR30 is anintracellular protein, found in the endoplasmic reticulum, which bindsestrogen with high affinity (K_(d) of 6 nM) and mediates rapid cellularresponses including calcium mobilization and phosphatidylinositol3,4,5-trisphosphate production in the nucleus. GPR30 receptor refers toall types of GPR30 receptor, regardless of the tissue in which suchreceptor is found and refers to any variant thereof, including receptorsof mammals (such as humans and domesticated mammals where veterinaryapplications are relevant) and variants thereof. Other names which havebeen used for GPR30 include CMKRL2, DRY12, FEG-1, GPCR-Br, LERGU,LERGU2, LyGPR, CEPR and MGC99678, among others.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent refers to, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression that can be usedto communicate the usefulness of a compound, composition, assay ormethod of the invention in a kit for suppressing or reducing systemicimmune response in a subject. The instructional material of the kit ofthe invention can, for example, be affixed to a container which containsthe identified compound, composition, assay, or methods of the inventionor be shipped together with a container that contains the identifiedcompound, composition, assay, or method. Alternatively, theinstructional material can be shipped separately from the container withthe intention that the instructional material and the compound,composition, assay, or method be used cooperatively by the recipient.

As used herein, the term “MC1R” refers to melanocortin receptor 1.

As used herein, the term “MITF” refers to microphthalmia-associatedtranscription factor.

As used herein, the term “modulate” means, with respect to diseasestates or conditions associated with binding of a compound of thepresent invention to a receptor contemplated in the present invention,to produce, either directly or indirectly, an improvement or lesseningof a condition or disease state which was, prior to administration of acompound according to the present invention, sub-optimal and in manycases, debilitating and even life threatening. Modulation may occur byvirtue of agonist activity, antagonist activity or mixedagonist/antagonist activity (depending on the receptor site).

As used herein, the term “P4” refers to progesterone.

“Parenteral” administration of a composition includes, e.g.,subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

As used herein, the term “PAQR7” refers to Progestin and AdipoQ Receptor7.

As used herein, the term “pharmaceutical composition” or “composition”refers to a mixture of at least one compound useful within the inventionwith other chemical components, such as carriers, stabilizers, diluents,dispersing agents, suspending agents, thickening agents, and/orexcipients. The pharmaceutical composition facilitates administration ofthe compound to an organism. Multiple techniques of administering acompound exist in the art including, but not limited to: intravenous,oral, aerosol, parenteral, ophthalmic, pulmonary, intracranial,transdermal and topical administration. In certain embodiments, theadministration comprises topical administration.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the composition, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the subject such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the subject. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar, buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the subject.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compound prepared from pharmaceuticallyacceptable non-toxic acids and/or bases, including inorganic acids,inorganic bases, organic acids, inorganic bases, solvates (includinghydrates) and clathrates thereof.

As used herein, a “pharmaceutically effective amount,” “therapeuticallyeffective amount” or “effective amount” of a compound is that amount ofcompound that is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

As used herein, the term “PR” refers to progesterone receptor.

The term “prevent,” “preventing” or “prevention” as used herein meansavoiding or delaying the onset of symptoms associated with a disease orcondition in a subject that has not developed such symptoms at the timethe administering of an agent or compound commences. Disease, conditionand disorder are used interchangeably herein.

By the term “specifically bind” or “specifically binds” as used hereinis meant that a first molecule preferentially binds to a second molecule(e.g., a particular receptor or enzyme), but does not necessarily bindonly to that second molecule.

As used herein, the terms “subject” and “individual” and “patient” canbe used interchangeably and may refer to a human or non-human mammal ora bird. Non-human mammals include, for example, livestock and pets, suchas ovine, bovine, porcine, canine, feline and murine mammals. In certainembodiments, the subject is human.

As used herein, the term “TMX” refers to tamoxifen.

As used herein, “topical administration” or “topical application” refersto a medication applied to body surfaces such as the skin or mucousmembranes.

The term to “treat,” as used herein, means reducing the frequency withwhich symptoms are experienced by a patient or subject or administeringan agent or compound to reduce the severity with which symptoms areexperienced. An appropriate therapeutic amount in any individual casemay be determined by one of ordinary skill in the art using routineexperimentation.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., acomposition useful within the invention (alone or in combination withanother pharmaceutical agent), to a subject, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a subject (e.g., for diagnosis or ex vivo applications), who has adisease or disorder, a symptom of a disease or disorder or the potentialto develop a disease or disorder, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease or disorder, the symptoms of the disease or disorder or thepotential to develop the disease or disorder. Such treatments may bespecifically tailored or modified, based on knowledge obtained from thefield of pharmacogenomics.

As used herein, the term “UV” refers to ultraviolet.

Throughout this disclosure, various aspects of the invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Compounds and Compositions

In one aspect, the present invention encompasses a compound that bindsto PAQR7, and does not bind to a canonical nuclear progesterone receptor(PR). In certain embodiments, the compound is a PAQR7 agonist.

In certain embodiments, the compound of the invention, or a salt,solvate, tautomer, enantiomer or diastereoisomer thereof, is CH2P4 (alsoknown as 19-CH2P4;rel-(8S,9S,10S,13S,14S,17S)-17-acetyl-13-methyl-10-vinyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3(2H)-one):

In another aspect, the present invention encompasses an estrogenderivative or analog thereof, that binds to GPER, such as but notlimited to GPER1, and does not bind to a canonical nuclear estrogenreceptor (ER). In certain embodiments, the estrogen derivative or analogis a GPER antagonist.

In certain embodiments, the compound of the invention, or a salt,solvate, tautomer, enantiomer or diastereoisomer thereof, is at leastone molecule of formula (I):

wherein: R₁ is selected from the group consisting of H and C₁-C₃ alkyl;R₂ is selected from the group consisting of H and C₁-C₃ alkyl; bond α isa single or double bond; R₃ is selected from the group consisting of Hand halo;

In certain embodiments, the compound is not G-15. In other embodiments,the compound is not G-36. In yet other embodiments, the compound is notG-15 or G-36.

In certain embodiments, R₁ is selected from the group consisting of H,methyl, ethyl, 1-propyl, and 2-propyl. In other embodiments, R₂ is H or2-propyl.

In certain embodiments, bond α is a double bond. In other embodiments,bond α is a single bond.

In certain embodiments, R₂ is selected from the group consisting of H,methyl, ethyl, 1-propyl and 2-propyl. In certain embodiments, R₂ is H.

In certain embodiments, R₃ is selected from the group consisting of H,F, Cl, Br and I. In certain embodiments, R₃ is Br.

In certain embodiments, the compound is at least one selected from thegroup consisting of:

In certain embodiments, the compound is at least one selected from thegroup consisting of:

G15 or G-15(rel-(3aS,4S,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline):

G36 or G-36(rel-(3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-8-isopropyl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolone):

rel-1-(2-((3aS,4S,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethyl)-3-(3-iodophenyl)urea(described in Ramesh, et al., 2010, J. Med. Chem. 53:1004-1014):

rel-(3aS,4S,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-8-((E)-1-(2-(5-iodopyridin-2-yl)hydrazono)ethyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline(described in Ramesh, et al., 2010, J. Med. Chem. 53:1004-1014):

In certain embodiments, the compound useful within the methods of theinvention, or a salt, solvate, enantiomer or diastereoisomer thereof, isat least one GPER antagonist recited in U.S. Patent ApplicationPublications No. 2008/0167334 and U.S. 2011/0092533, all of which areincorporated herein in their entireties by reference:

wherein:

X is ═N—, O, S, or N—R, with the proviso that when X is N—R and R is abond, N together with R¹ forms a 5- to 7-membered optionally substitutedheterocyclic group;

R is a bond, H, OH, NO₂, optionally substituted C₁-C₆ hydrocarbyl (suchas optionally substituted alkyl), optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocycle,optionally substituted —C(O)—(C₁-C₆) alkyl (amide), optionallysubstituted —C(O)—O—(C₁-C₆) alkyl (urethane), optionally substituted—C(O)—NH(C₁-C₆) alkyl (urea), optionally substituted—C(O)—N(C₁-C₆)dialkyl, optionally substituted —C(O)—NH(aryl), optionallysubstituted —C(O)—N(diaryl), optionally substituted—C(O)—NH(heteroaryl), optionally substituted —C(O)—N(diheteroaryl),optionally substituted —C(O)—NH(heterocycle) or optionally substituted—C(O)—N(diheterocycle);

R¹, R² and R⁵ are each independently selected from H, OH, NO₂, halogen,C₁-C₆ optionally substituted carboxylic acid group, optionallysubstituted O—(C₁-C₆)alkyl, optionally substituted C₁-C₆ hydrocarbyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocycle, optionally substituted —C(O)—(C₁-C₆)alkyl (ketone), optionally substituted —C(O)—O—(C₁-C₆) alkyl (ester),optionally substituted O—C(O)—(C₁-C₆) alkyl (ester), optionallysubstituted —C(O)—NH(C₁-C₆) alkyl (urea), optionally substituted—C(O)—N(C₁-C₆)dialkyl, optionally substituted —C(O)—NH(aryl), optionallysubstituted —C(O)—N(diaryl), optionally substituted—C(O)—NH(heteroaryl), optionally substituted —C(O)—N(diheteroaryl),optionally substituted —C(O)—NH(heterocycle) or optionally substituted—C(O)—N(diheterocycle);

R³ and R⁴ are each independently selected from H, OH, NO₂, halogen,C₁-C₆ optionally substituted carboxylic acid group, optionallysubstituted O—(C₁-C₆)alkyl, optionally substituted C₁-C₆ hydrocarbyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocycle, optionally substituted —C(O)—(C₁-C₆)alkyl (ketone), optionally substituted —C(O)—O—(C₁-C₆) alkyl (ester),optionally substituted O—C(O)—(C₁-C₆) alkyl (ester), optionallysubstituted —C(O)—NH(C₁-C₆) alkyl (urea), optionally substituted—C(O)—N(C₁-C₆)dialkyl, optionally substituted —C(O)—NH(aryl), optionallysubstituted —C(O)—N(diaryl), optionally substituted—C(O)—NH(heteroaryl), optionally substituted —C(O)—N(diheteroaryl),optionally substituted —C(O)—NH(heterocycle) or optionally substituted—C(O)—N(diheteroaryl) or R³ and R⁴ together form a 5- or 6-memberedoptionally substituted carbocyclic (which may be saturated orunsaturated), optionally substituted aryl, optionally substitutedheteroaryl or optionally substituted heterocyclic group;

R⁶ and R⁷ are each independently absent or are selected from H, OH, NO₂,halogen, C₁-C₆ optionally substituted carboxylic acid group, optionallysubstituted O—(C₁-C₆)alkyl, optionally substituted C₁-C₆ hydrocarbyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocycle, optionally substituted —C(O)—(C₁-C₆)alkyl (ketone), optionally substituted —C(O)—O—(C₁-C₆) alkyl (ester),optionally substituted O—C(O)—(C₁-C₆) alkyl (ester), optionallysubstituted —C(O)—NH(C₁-C₆) alkyl (urea), optionally substituted—C(O)—N(C₁-C₆)dialkyl, optionally substituted —C(O)—NH(aryl), optionallysubstituted —C(O)—N(diaryl), optionally substituted—C(O)—NH(heteroaryl), optionally substituted —C(O)—N(diheteroaryl),optionally substituted —C(O)—NH(heterocycle) or optionally substituted—C(O)—N(diheteroaryl), or together R⁶ and R⁷ form a 4-, 5-, 6- or7-membered optionally substituted carbocyclic (which may be saturated orunsaturated), optionally substituted aryl, optionally substitutedheteroaryl or optionally substituted heterocyclic group, or a 5- to9-membered optionally substituted carbocyclic or heterocyclic bicyclicgroup, with the proviso that R⁷ is not absent when both R^(7′) andR^(7″) are also absent;

R^(6′) is absent, H, C₁-C₆ optionally substituted hydrocarbyl group(such as H, CH₃ or CH₂CH₃) or together with R⁶ forms ═O;

R^(7′) is absent, H, optionally substituted hydrocarbyl group (such asH, CH₃ or CH₂CH₃), or together with R⁷ forms ═O;

R^(7″) is absent, H, OH, halogen, optionally substituted O—(C₁-C₆)alkyl,optionally substituted C₁-C₆ hydrocarbyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocycle,optionally substituted —C(O)—(C₁-C₆) alkyl (ketone), optionallysubstituted —C(O)—O—(C₁-C₆) alkyl (ester), optionally substitutedO—C(O)—(C₁-C₆) alkyl (ester), optionally substituted —C(O)—NH(C₁-C₆)alkyl (urea), optionally substituted —C(O)—N(C₁-C₆)dialkyl, optionallysubstituted —C(O)—NH(aryl), optionally substituted —C(O)—N(diaryl),optionally substituted —C(O)—NH(heteroaryl), optionally substituted—C(O)—N(diheteroaryl), optionally substituted —C(O)—NH(heterocycle) oroptionally substituted —C(O)—N(diheteroaryl);

R^(8′) is absent (when the carbon to which R^(8′) is attached and thecarbon to which R⁶ is attached form an optional double bond), H, CH₃ orCH₂CH₃;

R¹⁰, R¹¹, R¹² and R¹³ are each independently H, OH, NO₂, halogen, C₁-C₆optionally substituted carboxylic acid group, optionally substitutedO—(C₁-C₆)alkyl, optionally substituted C₁-C₆ hydrocarbyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted heterocycle, optionally substituted —C(O)—(C₁-C₆) alkyl(ketone), optionally substituted —C(O)—O—(C₁-C₆) alkyl (ester),optionally substituted O—C(O)—(C₁-C₆) alkyl (ester), optionallysubstituted —C(O)—NH(C₁-C₆) alkyl (urea), optionally substituted—C(O)—N(C₁-C₆)dialkyl, optionally substituted —C(O)—NH(aryl), optionallysubstituted —C(O)—N(diaryl), optionally substituted—C(O)—NH(heteroaryl), optionally substituted —C(O)—N(diheteroaryl),optionally substituted —C(O)—NH(heterocycle) or optionally substituted—C(O)—N(diheteroaryl);

R¹⁴ is H, OH, NO₂, halogen, C₁-C₆ optionally substituted carboxylic acidgroup, optionally substituted O—(C₁-C₆)alkyl, optionally substitutedC₁-C₆ hydrocarbyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocycle, optionally substituted—C(O)—(C₁-C₆) alkyl (ketone), optionally substituted —C(O)—O—(C₁-C₆)alkyl (ester), optionally substituted O—C(O)—(C₁-C₆) alkyl (ester),optionally substituted —C(O)—NH(C₁-C₆) alkyl (urea), optionallysubstituted —C(O)—N(C₁-C₆)dialkyl, optionally substituted—C(O)—NH(aryl), optionally substituted —C(O)—N(diaryl), optionallysubstituted —C(O)—NH(heteroaryl), optionally substituted—C(O)—N(diheteroaryl), optionally substituted —C(O)—NH(heterocycle) oroptionally substituted —C(O)—N(diheterocycle) or together with thecarbon to which R⁷ is attached forms a 5-, 6- or 7-membered optionallysubstituted carbocyclic (which may be saturated or unsaturated),optionally substituted aryl, optionally substituted heteroaryl oroptionally substituted heterocyclic ring;

Y is optionally substituted (CH₂)_(n) group where n is 0, 1 or 2,optionally substituted ═CH— group, a C═O group, O, S, optionallysubstituted N—(C₁-C₆)alkyl, optionally substituted N-aryl, optionallysubstituted N-heteroaryl, optionally substituted N-heterocycle,optionally substituted N—C(O)—(C₁-C₆)alkyl, optionally substitutedN—C(O)-aryl, optionally substituted N—C(O)-heteroaryl, optionallysubstituted N—C(O)-heterocycle;

Z is optionally substituted (CH₂)_(n) group where n is 1 or 2,optionally substituted ═CH— group, a C═O group, O, S, optionallysubstituted N—(C₁-C₆)alkyl, optionally substituted N-aryl, optionallysubstituted N-heteroaryl, optionally substituted N-heterocycle,optionally substituted N—C(O)—(C₁-C₆)alkyl, optionally substitutedN—C(O)-aryl, optionally substituted N—C(O)-heteroaryl, optionallysubstituted N—C(O)-heterocycle.

The compounds of the invention may possess one or more stereocenters,and each stereocenter may exist independently in either the (R) or (S)configuration. In certain embodiments, compounds described herein arepresent in optically active or racemic forms. The compounds describedherein encompass racemic, optically active, regioisomeric andstereoisomeric forms, or combinations thereof that possess thetherapeutically useful properties described herein. Preparation ofoptically active forms is achieved in any suitable manner, including byway of non-limiting example, by resolution of the racemic form withrecrystallization techniques, synthesis from optically active startingmaterials, chiral synthesis, or chromatographic separation using achiral stationary phase. A compound illustrated herein by the racemicformula further represents either of the two enantiomers or mixturesthereof, or in the case where two or more chiral center are present, alldiastereomers or mixtures thereof.

In certain embodiments, the compounds of the invention exist astautomers. All tautomers are included within the scope of the compoundsrecited herein.

Compounds described herein also include isotopically labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, 3H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³²P, and ³⁵S. In certain embodiments, substitution withheavier isotopes such as deuterium affords greater chemical stability.Isotopically labeled compounds are prepared by any suitable method or byprocesses using an appropriate isotopically labeled reagent in place ofthe non-labeled reagent otherwise employed.

In certain embodiments, the compounds described herein are labeled byother means, including, but not limited to, the use of chromophores orfluorescent moieties, bioluminescent labels, or chemiluminescent labels.

In all of the embodiments provided herein, examples of suitable optionalsubstituents are not intended to limit the scope of the claimedinvention. The compounds of the invention may contain any of thesubstituents, or combinations of substituents, provided herein.

In certain embodiments, the invention further provides pharmaceuticalcompositions comprising at least one compound of the invention and atleast one pharmaceutically acceptable carrier. In other embodiments, thepharmaceutical composition is formulated for topical and/or transdermalapplication. Topical formulations of the compounds contemplated withinthe invention may be used for skin lightening, as well as for treatingskin conditions or diseases associated with hyperpigmentation. Theseconditions or diseases include, but are not limited to, acne; eczema;chemical, sun, and thermal burn scars; lupus; psoriasis; sarcoidosis;pityriasis; erythema dyschromicum perstans; blistering diseases; drugreactions; lichen planus; or other inflammatory skin insults.

In certain embodiments, the invention provides topical formulationscomprising at least one sun-blocking agent and at least one compound ofthe invention. In other embodiments, the topical formulation comprises aformulated sunblock or sunscreen lotion and at least one compound of theinvention. In yet other embodiments, the at least one sun-blocking agentallows for protection against UV light damage caused by the natural sunlight, and the one or more compounds of the invention allow forlightening of the skin. In yet other embodiments, the melanin productiontriggered by the at least one compound of the invention further protectsthe skin against UV light damage caused by the natural sun light. In yetother embodiments, the topical formulation protects against UV-inducedskin damage and/or aging.

The present invention also pertains to kits useful within any of themethods of the invention described herein. Such kits comprise componentsuseful in any of the methods described herein, including for example,compositions and methods for modulating skin pigmentation in a mammaliansubject, such as a human, one or more containers (e.g., test tube, cellculture dish, cell culture plate, cell culture flask, cell culture bag)for containing a component of any of the embodiments of the inventiondescribed elsewhere herein, and instructional materials.

Salts

The compounds described herein may form salts with acids or bases, andsuch salts are included in the present invention. The term “salts”embraces addition salts of free acids or bases that are useful withinthe methods of the invention. The term “pharmaceutically acceptablesalt” refers to salts that possess toxicity profiles within a range thataffords utility in pharmaceutical applications. In certain embodiments,the salts are pharmaceutically acceptable salts. Pharmaceuticallyunacceptable salts may nonetheless possess properties such as highcrystallinity, which have utility in the practice of the presentinvention, such as for example utility in process of synthesis,purification or formulation of compounds useful within the methods ofthe invention.

Suitable pharmaceutically acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic,hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (includinghydrogen phosphate and dihydrogen phosphate). Appropriate organic acidsmay be selected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (or pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,sulfanilic, 2-hydroxyethanesulfonic, trifluoromethanesulfonic,p-toluenesulfonic, cyclohexylaminosulfonic, stearic, alginic,β-hydroxybutyric, salicylic, galactaric, galacturonic acid,glycerophosphonic acids and saccharin (e.g., saccharinate, saccharate).Salts may be comprised of a fraction of one, one or more than one molarequivalent of acid or base with respect to any compound of theinvention.

Suitable pharmaceutically acceptable base addition salts of compounds ofthe invention include, for example, ammonium salts and metallic saltsincluding alkali metal, alkaline earth metal and transition metal saltssuch as, for example, calcium, magnesium, potassium, sodium and zincsalts. Pharmaceutically acceptable base addition salts also includeorganic salts made from basic amines such as, for example,N,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (or N-methylglucamine) and procaine. All ofthese salts may be prepared from the corresponding compound by reacting,for example, the appropriate acid or base with the compound.

Methods

In one aspect, the invention includes a method of modulating skinpigmentation in a mammalian subject, such as a human. In certainembodiments, the method of the invention treats or prevents skinhyperpigmentation in the subject. In other embodiments, the method ofthe invention promotes skin lightening in the subject. In otherembodiments, the compound is administered topically or transdermally tothe subject.

In certain embodiments, the method comprises administering to thesubject a therapeutically effective amount of a compound that binds to anon-classical hormone receptor present on a melanocyte and does not bindto a canonical sex steroid hormone receptor, such as the estrogen and/orprogesterone receptor. In other embodiments, the non-classical hormonereceptor comprises PAQR7. In other embodiments, the non-classicalhormone receptor comprises GPER. In yet other embodiments, thenon-classical hormone receptor comprises GPER1 (also known as GPR30). Inyet other embodiments, the compound is a GPER antagonist and decreases,or prevents further gain of, skin pigmentation. In yet otherembodiments, the compound is a PAQR7 agonist and decreases, or preventsfurther gain of, skin pigmentation.

In certain embodiments, the subject is suffering from a conditioncomprising at least one selected from the group consisting of pigmentarychanges associated with oral contraceptive use, pregnancy, andendogenous estrogens in females (such as melasma and chloasma); solarlentigo; acne; eczema; chemical, sun, and thermal burn scars; lupus;psoriasis; sarcoidosis; pityriasis; erythema dyschromicum perstans;blistering diseases; drug reactions; lichen planus; or otherinflammatory skin insults.

Formulations/Administration

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated. By way of example, thecomposition may comprise between about 0.005% and about 100% (w/w) ofthe active agent, or any fractions or multiples thereof.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

In certain embodiments, the compositions comprising a compoundcontemplated within the invention are formulated using one or morepharmaceutically acceptable excipients or carriers. In certainembodiments, the pharmaceutical compositions of the invention comprise atherapeutically effective amount of at least one compound contemplatedwithin the invention and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers, which are useful, include, but arenot limited to, glycerol, water, saline, ethanol and otherpharmaceutically acceptable salt solutions such as phosphates and saltsof organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mack Publication Co., New Jersey).

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, are included in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. In certain embodiments, the administration comprisestopical administration. The pharmaceutical preparations may besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure buffers, coloring, flavoring and/oraromatic substances and the like. They may also be combined wheredesired with other active agents, e.g., other analgesic agents.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” that may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed. (1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of exposure to contaminants in theenvironment. Examples of preservatives useful in accordance with theinvention included but are not limited to those selected from the groupconsisting of benzyl alcohol, sorbic acid, parabens, imidurea andcombinations thereof. An illustrative preservative is a combination ofabout 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

The composition may include an antioxidant and a chelating agent thatinhibit the degradation of the compound. Selected antioxidants for somecompounds are BHT, BHA, α-tocopherol and ascorbic acid in the exemplaryrange of about 0.01% to 0.3%, such as BHT in the range of 0.03% to 0.1%by weight by total weight of the composition. For example, the chelatingagent is present in an amount of from 0.01% to 0.5% by weight by totalweight of the composition. Illustrative chelating agents include edetatesalts (e.g. disodium edetate) and citric acid in the weight range ofabout 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight bytotal weight of the composition. The chelating agent is useful forchelating metal ions in the composition which may be detrimental to theshelf life of the formulation. While BHT and disodium edetate areillustrative antioxidant and chelating agent respectively for somecompounds, other suitable and equivalent antioxidants and chelatingagents may be substituted as would be known to those skilled in the art.

The composition comprising a compound contemplated within the inventioncan be administered to a mammal as frequently as several times daily, orit may be administered less frequently, such as once a day, once a week,once every two weeks, once a month, or even less frequently, such asonce every several months or even once a year or less.

It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 0.5-5 mg per day dose may beinitiated on Monday with a first subsequent 0.5-5 mg per day doseadministered on Wednesday, a second subsequent 0.5-5 mg per day doseadministered on Friday, and so on. The frequency of the dose will bereadily apparent to the skilled artisan and will depend upon any numberof factors, such as, but not limited to, the type and severity of thedisease being treated, the type and age of the animal, and so forth.

Topical Administration

An obstacle for topical administration of pharmaceuticals is the stratumcorneum layer of the epidermis. The stratum corneum is a highlyresistant layer comprised of protein, cholesterol, sphingolipids, freefatty acids and various other lipids, and includes cornified and livingcells. One of the factors that limit the penetration rate (flux) of acompound through the stratum corneum is the amount of the activesubstance that can be loaded or applied onto the skin surface. Thegreater the amount of active substance applied per unit of area of theskin, the greater the concentration gradient between the skin surfaceand the lower layers of the skin, and in turn the greater the diffusionforce of the active substance through the skin. Therefore, a formulationcontaining a greater concentration of the active substance is morelikely to result in penetration of the active substance through theskin, and more of it, and at a more consistent rate, than a formulationhaving a lesser concentration, all other things being equal.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions. Such formulations maybe applied to the skin directly or through the use of swabs,applicators, spatulas and the like, as well as in the form oftransdermal patches. In certain embodiments, the patch minimizes loss ofpharmaceuticals through washing, friction, scratching and/or rubbing ofthe skin. In other embodiments, the patch increases absorption of thepharmaceutical through the skin, while minimizing the exposure of theskin to the pharmaceutical.

Topically administrable formulations may, for example, comprise fromabout 0.005% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

Enhancers of permeation may be used. These materials increase the rateof penetration of drugs across the skin. Typical enhancers in the artinclude ethanol, glycerol monolaurate, PGML (polyethylene glycolmonolaurate), dimethylsulfoxide, and the like. Other enhancers includeoleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylicacids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositionsof the invention may contain liposomes. The composition of the liposomesand their use are known in the art (for example, U.S. Pat. No.6,323,219).

In alternative embodiments, the pharmaceutical composition of theinvention may be optionally combined with other ingredients such asadjuvants, anti-oxidants, chelating agents, surfactants, foaming agents,wetting agents, emulsifying agents, viscosifiers, buffering agents,preservatives, and the like. In other embodiments, a permeation orpenetration enhancer is included in the composition and is effective inimproving the percutaneous penetration of the active ingredient into andthrough the stratum corneum with respect to a composition lacking thepermeation enhancer. Various permeation enhancers, including oleic acid,oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids,dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known tothose of skill in the art. In another aspect, the composition mayfurther comprise a hydrotropic agent, which functions to increasedisorder in the structure of the stratum corneum, and thus allowsincreased transport across the stratum corneum. Various hydrotropicagents such as isopropyl alcohol, propylene glycol, or sodium xylenesulfonate, are known to those of skill in the art.

The topically active pharmaceutical composition of the invention shouldbe applied in an amount effective to affect desired changes. As usedherein, “amount effective” shall mean an amount sufficient to cover theregion of skin surface where a change is desired. An active compoundshould be present in the amount of from about 0.0001% to about 15% byweight volume of the composition; or in an amount from about 0.0005% toabout 5% of the composition; or in an amount of from about 0.005% toabout 1% of the composition. Such compounds may be synthetically—ornaturally derived.

Controlled Release Formulations and Drug Delivery Systems

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.In some cases, the dosage forms to be used can be provided as slow orcontrolled-release of one or more active ingredients therein using, forexample, hydropropylmethyl cellulose, other polymer matrices, gels,permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, or microspheres or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the pharmaceutical compositions of the invention. Thus, single unitdosage forms suitable for topical administration, such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, transdermal patches, and solutions or suspensionsthat are adapted for controlled-release are encompassed by the presentinvention.

Most controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood level of the drug, andthus can affect the occurrence of side effects.

Most controlled-release formulations are designed to initially releasean amount of drug that promptly produces the desired therapeutic effect,and gradually and continually release of other amounts of drug tomaintain this level of therapeutic effect over an extended period oftime. In order to maintain this constant level of drug in the body, thedrug must be released from the dosage form at a rate that will replacethe amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by variousinducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. The term “controlled-releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, or microspheresor a combination thereof that facilitates the controlled-release of theactive ingredient.

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release that is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material which provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In certain embodiments of the invention, the compounds of the inventionare administered to a patient, alone or in combination with anotherpharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that may,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Materials and Methods

Unless otherwise noted, all cell lines, biological materials, chemicalmaterials, reagents and resins were obtained from commercial suppliersand used without purification.

Melanocyte Culture

Primary melanocytes were extracted from fresh discarded surgical humanforeskin specimens. After overnight incubation in Dispase, the epidermiswas separated from the dermis and treated with trypsin for 10 minutes.Cells were pelleted and plated on selective MC Medium 254 (Invitrogen)with Human Melanocyte Growth Supplement, and 1% penicillin andstreptomycin. Lightly pigmented primary melanocytes were utilized forexperiments assaying estrogen and GPER agonist effects, and heavilypigmented primary melanocytes were utilized for experiments assayingprogesterone and PAQR7 effects in melanin production. Progesterone(P8783), 17β-Estradiol (E8875), and αMSH (M4135) were purchased fromSigma-Aldrich. G-15 (14673) and G-36 (14397) were purchased from CaymanChemical. CH2P4 (2085) was purchased from Axon Medchem. Pertussis toxinwas purchased from R&D systems (3097). These compounds were diluted toworking stock solutions in Medium 254.

Melanin Assay

2×10⁵ melanocytes were seeded uniformly on 6-well tissue culture plates.Cells were treated with vehicle controls, sex steroids, hormonederivatives, or pertussis toxin for 4 days. Cells were then trypsinized,counted, and spun down at 300×g for 5 minutes. The resulting cell pelletwas solubilized in 1M NaOH, and boiled for 5 minutes. The opticaldensity of the resulting solution was read at 450 nm using an EMaxmicroplate reader (Molecular Devices). The absorbance was normalized tothe number of cells in each sample, and relative amounts of melanin wereset based on vehicle treated controls. For tissue melanin assays, tissuewas weighed prior to boiling in 1M NaOH for 20 minutes. Samples werespun down to eliminate insoluble materials, and then the optical densityof the sample was measured and normalized to the weight of tissue.

cAMP ELISA

cAMP ELISA was performed on primary human melanocytes using the CyclicAMP XP Assay Kit (Cell Signaling Technology, #4339) followingmanufacturer instructions.

Western Blot Analyses and Antibodies

Adherent cells were treated with 1 μM doses of E2 and P4 overnight,washed once with DPBS, and lysed with 1% NP-40 buffer (150 mM NaCl, 50mM Tris, pH 7.5, 1 mM EDTA, and 1% NP-40) containing 1× proteaseinhibitors (Roche) and 1× phosphatase inhibitors (Roche). Lysates werequantified (Bradford assay), normalized, reduced, denatured (95° C.) andresolved by SDS gel electrophoresis on 4-15% Tris/Glycine gels(Bio-Rad). Resolved protein was transferred to PVDF membranes(Millipore) using a Semi-Dry Transfer Cell (Bio-Rad), blocked in 5% drymilk in TBS-T and probed with primary antibodies recognizing MITF (CellSignaling Technology, #12590, 1:1000), pCREB (Cell Signaling Technology,#9198, 1:1000), CREB (Cell Signaling Technology, #9104, 1:1000), andβ-Actin (Cell Signaling Technology, #3700, 1:4000). After incubationwith the appropriate secondary antibody, proteins were detected usingeither Luminata Crescendo Western HRP Substrate (Millipore) or ECLWestern Blotting Analysis System (GE Healthcare).

Melanin Staining

Formalin-fixed paraffin embedded tissue was sectioned at 5 uM andcollected on superfrost plus slides (Fisher), and subjected toFontana-Masson stain for melanin. Briefly, sections were deparaffinized,rehydrated, and incubated in the following solutions: 2.5% aqueoussilver nitrate for 10 min, 0.1% aqueous gold chloride for 15 min, and 5%aqueous sodium thiosulfate for 5 min. Distilled deionized water was usedfor rinsing and incubations were done at room temperature except forsilver nitrate at 60° C. Slides were counterstained with 0.1% nuclearfast red Kernechtrot for 5 min, dehydrated, cleared, and coverslippedusing MM24 mounting media (Leica). All staining reagents were fromPolyscientific R & D Corporation.

Immunohistochemistry

Formalin fixed paraffin embedded (FFPE) human skin tissue sections fromorganotypic tissue was stained for MITF protein expression using aprimary antibody to MITF (Leica Biosystems, NCL-L-MITF, 1:15). Stainingwas performed following the manufacturer protocol for high temperatureantigen unmasking technique for immunohistochemical demonstration onparaffin sections.

Quantification of Melanin Staining

Tissue sections from organotypic culture were stained using methodsdescribed elsewhere herein. In terms of quantitation, briefly, 20×photomicrograph images of representative tissue sections were takenusing the Zeiss Axiophot microscope. Tiff files of the images were savedand transferred to Adobe PHOTOSHOP® where pixels corresponding toFontana-Masson staining and epidermal counter stain were selected usingthe color selection tool. Images corresponding to the single specificcolor were then analyzed using FIJI (Image J) to determine the number ofpixels in each sample. The numbers of pixels representing Fontana-Massonstaining were normalized to the total amount epidermal counter staining.Final ratios Fontana-Masson staining in the epidermis were set relativeto amount of staining in vehicle treated controls.

Quantitative RT/PCR

mRNA was extracted from melanocytes according to the RNeasy Mini Kitprotocol (Qiagen), and reverse transcribed to cDNA using the HighCapacity RNA-to cDNA kit (Applied Biosystems). Quantitative PCR of theresulting cDNA was carried out using Power SYBR Green Master Mix(Applied Biosystems) and gene-specific primers, in triplicate, on a ViiA7 Real-Time PCR System (Life Technologies).

The following primers were used for detection; B-Actin forward: 5′-CATGTA CGT TGC TAT CCA GGC-3′ (SEQ ID NO:1); B-Actin reverse: 5′-CTC CTTAAT GTC ACG CAC GAT-3′ (SEQ ID NO:2); ER-A forward: 5′-AAA GGT GGG ATACGA AAA GAC C-3′ (SEQ ID NO:3); ER-A reverse: 5′-AGC ATC CAA CAA GGC ACTGA-3′ (SEQ ID NO:4); ER-B forward: 5′-GGC TGC GAG AAA TAA CTG CC-3′ (SEQID NO:5); ER-B reverse: 5′-AAT GCG GAC ACG TGC TTT TC-3′ (SEQ ID NO:6);PGR forward: 5′-AGG TCT ACC CGC CCT ATC TC-3′ (SEQ ID NO:7); PGRreverse: 5′-AGT AGT TGT GCT GCC CTT CC-3′ (SEQ ID NO:8); AR forward:5′-GTG CTG TAC AGG AGC CGA AG-3′ (SEQ ID NO:9); AR reverse: 5′-GTC AGTCCT ACC AGG CAC TT-3′ (SEQ ID NO:10); GPER forward: 5′-ACA GAG GGA AAACGA CAC CT-3′ (SEQ ID NO:11); GPER reverse: 5′-AAT TTT CAC TCG CCG CTTCG-3′ (SEQ ID NO:12); PAQR7 forward: 5′-GTG CAC TTT TAT ACC GTC TGCTT-3′ (SEQ ID NO:13); PAQR7 reverse: 5′-CCT GGG CAG GGA GCTAAG AT-3′(SEQ ID NO:14). Relative expression was determined using the2-[delta][delta] Ct method followed by normalization to the AR receptortranscript levels in MCF7 cells.

Lentiviral Victors

The following shRNAs were expressed from the GIPZ vector (OpenBiosystems): shPAR7.3 (V3LHS_364596, 5′-TGT GGT AGA GAA GAG CTG G-3′;SEQ ID NO:15), shPAQR7.4 (V3LHS_364598, AGAAGTGTGCCAAGGCACT; SEQ IDNO:16) shGPER.1 (V2LHS_132008, 5′-TCC TTC TCC TCT TTA ACT C-3′; SEQ IDNO:17), shGPER.3 (V3LHS_390319, 5′-TGA TGA AGT ACA GGT CGG G-3′; SEQ IDNO:18).

Guide RNAs were designed using software tools provided on the websitewww dot genome-engineering dot org/. Guide RNAs were subsequently clonedinto lentiCRISPRv2 (Addgene #52961) according to the accompanyingprotocol. Guide RNA sequences are as follows: lentiCRISPR GFP, 5′-GAAGTT CGA GGG CGA CAC CC-3′ (SEQ ID NO:19); lentiCRISPR GPER.1,5′-ACAGGCCGATCACGTACTGC-3′ (SEQ ID NO:20); lentiCRISPR GPER.2, 5′-GAGCAC CAG CAG TAC GTG AT-3′ (SEQ ID NO:21); lentiCRISPR PAQR7.1, 5′ CGTACATCT ATG CGG GCT AC-3′ (SEQ ID NO:22); lentiCRISPR PAQR7.5, 5′-CGT GCGGAA ATA GAA GCG CC-3′ (SEQ ID NO:23).

Non-Limiting Exemplary Topical Treatment

2% (w/v) compound is prepared in DMSO. 20 μL of this solution is applieddaily to the right ear, with vehicle only applied to the left ear, of4-week-old C57BL/6 mice. These studies are performed withoutinclusion/exclusion criteria, randomization, or blinding. Based on atwo-fold anticipated effect, this experiment is performed with 3biological replicates.

Statistical Analysis

* denotes a P-value of less than 0.05 in an unpaired, two-tailedStudents T-Test.

Example 1 Preparation of 3-D Organotypic Skin Cultures

Organotypic skin grafts containing MCs were established usingmodifications to previously detailed methods (Ridky, et al., Nature Med.2010, 16, 1450-1455; Chudnovsky, et al., Nature Gen. 2005, 37, 745-749).The Keratinocyte Growth Media (KGM) used for keratinocyte-only skingrafts was replaced with modified Melanocyte Xenograft Seeding Media(MXSM). MXSM is a 1:1 mixture of KGM, lacking cholera toxin, andKeratinocyte Media 50/50 (Gibco) containing 2% FBS, 1.2 mM calciumchloride, 100 nM Et-3 (endothelin 3), 10 ng/mL rhSCF (recombinant humanstem cell factor), and 4.5 ng/mL r-basic FGF (recombinant basicfibroblast growth factor). 1.5×10⁵ melanocytes and 5.0×10⁵ keratinocyteswere suspended in 80 μL MXSM, seeded onto the dermis, and incubated at37° C. for 8 days at the air-liquid interface.

Example 2 Estrogen and Progesterone Reciprocally Regulate MelaninSynthesis

To examine whether sex steroids directly influence melanin synthesis,normal primary human melanocytes was treated with estrogen(17β-estradiol), which resulted in a dose dependent increase in melaninproduction (FIG. 2C). 25 nM estrogen were used for subsequentexperiments, a medically-relevant concentration observed during secondand third trimester pregnancy. After 4 days, melanin production wasmarkedly increased (208±27%) (FIG. 3A). The change in melanin productionwas similar in magnitude to changes observed with αMSH (FIGS. 2B & 2D).This melanin-promoting estrogen effect was consistent with involvementof estrogen in melanin synthesis. To examine the effects of estrogen onmelanocyte homeostasis in the context of intact human epidermis,architecturally-faithful 3-D organotypic skin was established utilizingnormal primary epidermal keratinocytes and melanocytes within theframework of native stroma and intact basement membrane. After 1 week,estrogen-treated skin displayed a 3-fold increase in melanin content(FIG. 3B), without any increase in total melanocyte number or density(FIG. 3C).

Physiologic estrogen effects in other tissue types are oftencounter-balanced by simultaneous exposure to progesterone, another sexsteroid that increases markedly during pregnancy. To determine whetherthis reciprocal relationship is also active in melanocytes, cells weretreated with physiologic levels of progesterone, which resulted in adose dependent decrease in melanocyte melanin production (FIG. 2E). Aconcentration of 500 nM was used for subsequent experiments, a levelreflective of that seen during 3^(rd) trimester pregnancy. Progesteronedecreased melanin production by nearly half (58±11.4%), both in culture(FIG. 3D) and in skin tissue (FIG. 3E), without affecting overallmelanocyte cell number (FIG. 3F).

To determine whether female cells also responded similarly, femaleiPS-derived melanocytes were treated with estrogen and progesterone.Responses similar to those observed with the male cells were noted (FIG.2F).

To determine whether melanocytes isolated from body sites other thanforeskin also responded similarly to sex hormones, melanocytes fromadult facial skin were treated with estrogen and progesterone; responsesobserved were similar to those observed with the foreskin melanocytes(FIG. 2G).

Example 3 Primary Human Melanocytes do not Express Nuclear Estrogen orProgesterone Receptors (ER/PR), and Respond to Sex Steroids Via AlteredcAMP Signaling

To determine the mechanisms through which estrogen and progesteronemediate their pigment effects, components of the canonical pigmentproduction pathway were examined. An increase in cAMP upon estrogentreatment (FIG. 4A) was observed. This suggests that estrogen accessesthe canonical pigment production pathway downstream of MC1R. Consistentwith this, pCREB and MITF proteins were similarly induced (FIG. 4B). Incontrast, progesterone treatment resulted in diminished pigmentproduction, with corresponding decreases in cAMP, pCREB and MITF (FIGS.4C-4D).

Without wishing to be limited by any theory, these data suggest thatestrogen, progesterone, and α-MSH converge on the canonical melaninproduction pathway at the level of adenylate cyclase to reciprocallymodulate melanin synthesis. Consistently, the estrogen pigment effectswere significantly attenuated in the presence of physiologicprogesterone (FIG. 4E). This likely explains why pregnancy-associatedhyperpigmentation is not observed all over the body, but ischaracteristically limited to specific areas where melanocyte density orUV radiation exposure is highest, including the face, genital, andareolar regions.

As steroid hormones are not predicted to signal through MC1R, whichnatural ligand is the peptide hormone αMSH, efforts were put forth toidentify the specific receptors through which estrogen and progesteronemediate their effects on melanin synthesis. Classical estrogen (ER) andprogesterone (PR) receptors were not detected in melanocytes usingqRT-PCR, despite robust expression in the MCF7 ER/PR positive breastcell line (FIG. 5A). RNAseq studies in primary human melanocytes failedto detect ER or PR transcripts (FIG. 5B). No ER or PR protein wasdetected via western blotting of melanocyte protein lysates, althoughboth receptors were readily apparent in MCF7 extracts (FIG. 4F).

Example 4 Sex Steroid Signaling in Melanocytes is Dependent on theNonclassical Membrane Bound G-Protein Coupled Sex Hormone Receptor GPER

Since MC1R is a G protein-coupled receptor (GPCR), it was hypothesizedthat an alternative GPCR may be binding sex hormones to mediate pigmenteffects. To identify possible candidates, whole transcriptome RNAseqdata from primary human MCs to identify other expressed GPCRs wereanalyzed. The membrane-bound, G protein-coupled estrogen receptor (GPER)was expressed (FIG. 5B). An analoguous, noncanonical G protein-coupledprogesterone receptor, porogestin and adipoQ receptor 7, was alsodetected (FIG. 5B). Primary human MCs were analyzed for expression ofGPER and PAQR7 by qRT-PCR, and their expressions were verified (FIG.5C). Notably, GPER and PAQR7 expressions were markedly lower in otherskin cell types including keratinocytes and fibroblasts (FIG. 5D).

shRNA-mediated knockdown was utilized to establish the necessity of GPERand PAQR7 to mediate sex hormone effects in MCs (FIGS. 5E-5F). GPER orPAQR7 depletion using either of two independent hairpins completelyeliminated the melanocyte pigmentation response to estrogen andprogesterone, respectively (FIGS. 4G-4H).

To verify these results, a complementary genetic approach based onCRISPR-Cas9 mediated gene disruption of GPER or PAQR7 was used, whichalso completely blocked the pigmentary response to estrogen andprogesterone, respectively (FIGS. 5G-5H).

PAQR7 binds progesterone and regulates the final stages of sea troutoocyte meiosis through cAMP reduction. In that fish system, PAQR7signals through G protein complexes containing the inhibitory G subunit(G_(i)). G_(i) subunits function to repress adenylate cyclase, resultingin a decrease in cytosolic cAMP. To examine whether this signalingmechanism was also functional and relevant in mammals, primary humanmelanocytes were treated with progesterone in the presence of pertussistoxin (PTX), an exotoxin that catalyzes the ADP-ribosylation of G_(i)subunits. This modification prevents G_(i) from interacting with GPCRs,and therefore prevents their activation upon GPCR ligand binding.Treatment of melanocytes with PTX completely blocked progesteronepigment effects, establishing that progesterone signals through G_(i)subunits in melanocytes (FIG. 6). The finding that PAQR7 works throughinhibitory G_(i) subunits is interesting, as it the first example of amelanocyte cellular signaling cascade that actively represses melaninsynthesis at the level of G-protein signaling, as opposed to classicallydefined pigment control mechanisms that modulate the strength of thestimulatory MC1R signal. In many animal systems, the Agouti proteindecreases pigment production via physically binding to MC1R andinhibiting αMSH stimulation, rather than through an actively suppressivemechanism.

Example 5 Specific Activation of GPER Alters Pigment Production in HumanSkin Tissue

To complement these genetic studies establishing that estrogen andprogesterone pigment effects are mediated through nonclassical sexsteroid receptors, small molecule steroid analogs were utilized withspecific agonist activity on ER, PR, GPER or PQAR7. Tamoxifen, an ERantagonist, is associated with development of melasma in breast cancerpatients. The mechanistic basis for this clinical observation waspreviously unknown and somewhat perplexing, given that tamoxifen blocksestrogen receptor function and thus might have been predicted to limitmelanin synthesis. However, tamoxifen acts as a GPER agonist. Tamoxifentreatment resulted in a dose-dependent increase in melanin production tolevels comparable to those observed with native estrogen (FIG. 7A),consistent with the model in which GPER activation promotes melaninsynthesis. Most oral contraceptives incorporate an estrogen derivative(ethinyl estradiol) for the estrogen component. Treatment of primaryhuman MCs with ethinyl estradiol also resulted in a dose dependentincrease in melanin production, indistinguishable from native estrogenat an equal concentration (FIG. 7B).

Further establishing that GPER is the melanocyte estrogen receptor, aGPER agonist (G-1;rel-1-[4-(6-bromo-1,3-benzodioxol-5-yl)-3aR,4S,5,9bS-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone)and estrogen effects were blocked by either of two specific GPERantagonists, G-15 and G-36 (FIG. 8), which do not have inhibitoryactivity against ER. To establish that PAQR7 signaling is sufficient todecrease melanin production, a specific PAQR7 agonist Org OD-02 (CH2P4),which does not bind PR, was used. CH2P4 caused a dose-dependent,PAQR7-dependent, decrease in melanin production (FIGS. 9A-9B and10A-10C).

Example 8 In Vivo Studies

Tissue generated elsewhere herein is orthotopically xenografted ontoimmunocompromised mice, allowing analysis of human skin in vivo.Hormones are administered subcutaneously or topically to demonstrate thenecessity of GPER to mediate hormone-induced changes in pigmentation.The effects of the specific agonists are evaluated.

To demonstrate that this mechanism is not restricted to foreskin-derivedmelanocytes, melanocytes are isolated from various body sites and donorsof different age, sex, and race. 3-D organotypic cultures areestablished with melanocytes from these sources, and the necessity andsufficiency of GPER signaling is evaluated.

Example 9 Evaluation of Mechanism Through which GPER InfluencesPigmentation

To analyze the mechanism through which estrogen and progesteroneinfluence pigmentation downstream of GPER, the Gα subunit activated bythe receptor is identified. To accomplish this, agonist-promoted[³⁵S]GTPγS exchange assays are used, followed by immuno-enrichment ofspecific G protein subgroups. G protein-coupled receptor signalingdrives guanine nucleotide exchange on the Gα subunit, switching GDP forGTP. [³⁵S]GTPγS is an isotopic, non-hydrolysable analogue of GTP.Membrane preparations from primary human melanocytes are isolated andstimulated with GPER agonist, as well as vehicle controls, in thepresence of [³⁵S]GTPγS. Next, Gs and Gi subunits are immunoprecipitatedfrom the samples, and [³⁵S]GTPγS binding is measured using ascintillation spectrophotometer. Melanocytes with ablated GPER are usedas negative controls in these assays.

Both the Gs and Gi subunits influence adenylate cyclase activity, whichfunctions to produce cAMP. To examine the mechanism through which GPERis regulating the canonical pigmentation pathway downstream of G proteinsignaling, adenylate cyclase activity is analyzed in the presence ofestrogen and progesterone using cAMP ELISA, relative to vehicle-treatedcontrols. Western analysis is used to examine downstream changes inpCREB and MITF relative to vehicle-treated controls.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed:
 1. A method of decreasing or reversing gain of skinpigmentation in a mammalian subject, the method comprising: topically ortransdermally administering to the subject a therapeutically effectiveamount of at least one compound of Formula (I), or a salt, solvate,tautomer, enantiomer, or diastereomer thereof:

wherein: R₁ is selected from the group consisting of H and C₁-C₃ alkyl;R₂ is selected from the group consisting of H and C₁-C₃ alkyl; bond a isa single or double bond; and R₃ is selected from the group consisting ofH and halo; with the proviso that the compound is not

and wherein the compound does not bind to a canonical estrogen receptor(ER) in the subject.
 2. The method of claim 1, wherein the compound is aGPER1 antagonist.
 3. The method of claim 1, wherein the subject issuffering from at least one condition selected from the group consistingof pigmentary changes associated with oral contraceptive use, pregnancy,and endogenous estrogens in females; solar lentigo; acne; eczema;chemical, sun, and thermal burn scars; lupus; psoriasis; sarcoidosis;pityriasis; erythema dyschromicum perstans; blistering diseases; drugreactions; and lichen planus.
 4. The method of claim 1, wherein thecompound is formulated as a pharmaceutical composition for topical ortransdermal administration.
 5. The method of claim 4, wherein thepharmaceutical composition further comprises at least one selected fromthe group consisting of a sun-blocking agent and a sunscreen lotion. 6.The method of claim 4, wherein the pharmaceutical composition isessentially free of a skin bleaching agent.
 7. The method of claim 1,wherein the compound is at least one selected from the group consistingof: